Recently evidence has been obtained implicating several GABAA receptor and protein kinase genes as potentially important for the actions of ethanol at the GABAA receptor. For example, the gamma21 subunit of the receptor has been shown to be necessary for ethanol to exert its potentiating effect on the GABAA receptor. In addition, it appears that phosphorylation of this subunit, presumably by protein kinase C, is required to generate this effect. Therefore, the objective of this component will be to use a number of specific molecular biological approaches to determine whether there are changes in the sequence and/or expression in these genes in lines of mice that exhibit differential responsiveness to alcohol. Changes which influence the amino acid sequence of these genes will be sought by direct DNA sequencing of PCR- amplified DNA corresponding to the protein coding regions of selected GABAA receptor and protein kinase genes from lines of mice known to genetically differ in ethanol sensitivity, dependence, and preference. Changes in the expression of these genes in specific brain regions will be investigated by Northern analysis, in situ hybridization, Western analysis and quantitative receptor autoradiography. When changes in the structure and/or expression of any of these genes are identified, the extent to which a specific change correlates with a particular alcohol- related phenotype will be determined. Molecular genetic differences found to exhibit a significant correlation with an alcohol-related phenotype will provide a candidate gene (or genes) for genetic predisposition to alcoholism which could then be tested in human genetic studies.